The invention relates to an optical device with a focusing optic that focuses a light beam in a focal plane, and with a means of forming the focus of the light beam, whereby the means of forming a focus causes a phase shift between a first part of the light beam and a second part of the light beam.
The invention furthermore relates to a scanning microscope with a focusing optic that focuses a light beam in a focal plane, and with a means of forming the focus of the light beam, whereby the means of forming the focus of causes a phase shift between a first part of the light beam and a second part of the light beam.
The invention furthermore relates to a phase filter that brings about a phase shift between a first part of a light beam and a second part of a light beam.
In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflection light or fluorescent light emitted by the sample. The focus of the illumination light beam is moved in an object plane with the help of a controllable beam deflector, generally by tipping two mirrors, whereby the axes of deflection are mostly positioned perpendicular to each other so that one mirror deflects in the x-direction and the other in the y-direction. The mirrors can, for example, be tipped with the help of galvanometric positioners. Measurement of the power of the light coming from the object is dependent on the position of the scanning beam. Usually, the positioners are equipped with sensors that determine the current position of the mirrors. In addition to these so-called beam-scanning methods, scanning microscopes with spatially fixed illumination light beams are also known, in which the sample is scanned with the help of a fine-positioning stage. Such scanning microscopes are referred to as object-scanning microscopes.
In confocal scanning microscopy in particular, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optic with which the light from the source is focused on a pinhole aperture—the so-called excitation aperture—, a beam splitter, a beam deflector to control the beam, a microscope optic, a detection aperture, and detectors to detect the detection light or fluorescent light. The illumination light is coupled via a beam splitter. The fluorescent light or reflection light coming from the object returns to the beam splitter via the beam reflector, passes this, and is subsequently focused on the detection aperture, behind which the detectors are located. Detection light that does not originate directly from the focal region takes another light path and does not pass through the detection aperture so that one obtains point information by which a three-dimensional image is constructed by sequential scanning of the object. In most cases, a three-dimensional image is achieved by layered imaging.
The power of the light coming from the object is measured at set intervals during the scanning process so that it is scanned pixel by pixel. Each reading must be definitively assigned to the corresponding scanning position so that an image can be created from the measured data. Advantageously, the status data of the beam deflector positioners are measured continually, or the target control data from the beam deflector are used directly, which is, however, not as precise.
It is also possible to detect, for example, the fluorescent light or the transmission of the excitation light from the condenser in a transillumination configuration. The detection light beam then does not reach the detector via the scanning mirror (non-descanning configuration). In the transillumination configuration, a detection aperture at the condenser would be necessary to detect fluorescent light in order to achieve three-dimensional resolution as in the described descanning configuration. In the case of two-photon excitation, one can forgo the detection aperture at the condenser because the excitation probability is dependent on the square of the photon density (proportional to the intensity squared), which is naturally much higher in the focal region than in neighboring regions. The detectable fluorescent light therefore originates almost entirely and with great certainty from the focal region, which makes it superfluous to differentiate fluorescent photons from the focal region from fluorescent photons from the neighboring regions using an aperture arrangement.
The resolution of a confocal scanning microscope is, among other things, dependent on the intensity distribution and the spatial spread of the focus of the excitation light beam. An arrangement to increase the resolution for fluorescent uses is known from PCT/DE/95/00124. Here, the lateral marginal regions of the focal volume of the excitation light beam are illuminated with a light beam of another wavelength—the so-called stimulation light beam, which is emitted by a second laser—in order to return the sample regions excited by the light from the first laser to their ground state stimulated. What is then detected is only the spontaneously emitted light from the regions that were not illuminated by the second laser, so that an overall improvement in resolution is achieved. This method has come to be known as STED (Stimulated Emission Depletion).
A new development has demonstrated that one can achieve both lateral and axial improvements in resolution if one succeeds in hollowing the inside of the focus of the stimulation light beam. To this end, a phase filter that contains a round λ/2 plate that is smaller in diameter than the diameter of the beam is introduced into the beam path of the stimulation light beam and is over-illuminated as a result. The form of the stimulation focus results from the Fourier transforms of the phase filter function.
Currently in STED microscopy, a sample is illuminated with a shortwave laser pulse which, for example, comes from a pulsed laser diode. Then, a longwave laser pulse, which has a high intensity, causes a de-excitation of the fluorescent molecules at the margin of the excitation focus. In order to minimize as much as possible the photodamaging effect of the longwave pulse, it should have a pulse width in the range of approximately 100 ps or longer. This longwave laser pulse can, for example, be emitted by a second laser diode that already has the appropriate pulse width, or it may be generated by broadening the pulse of a short longwave laser pulse. A pulse can be most easily broadened by a two-step process. First, the short pulse passes through a glass rod, which stretches it to a pulse width of several picoseconds. After that, the laser pulse passes through a long glass fiber in which it is broadened further. A glass rod can also be replaced by a glass body in which the pulse is multiply reflected by total reflection and therefore travels a long distance within the glass body. Another way of stretching the laser pulse is to use diffraction grating.
In order to de-excite the fluorescence molecules at the focal margin, the de-excitation point spread function must be specially formed. To this end, phase filters are used that are located in the beam path of the longwave laser beam. They change the wave front of the de-excitation beam depending on where it is located. Phase filters are currently manufactured by applying a transparent structure (e.g., magnesium fluoride) to a glass substrate. The light that penetrates the structure and the glass substrate experiences a phase delay in comparison to the light that only penetrates the substrate that is dependent on the thickness of the structure and the wavelength of the light. When using different wavelengths, as is, for example, the case when using different fluorescent dyes, a different phase filter must be used for each wavelength. The disadvantage is that when using a broad wavelength spectrum to de-excite, as is the case when broadening the pulse in a glass fiber or when using photonic crystal fibers (photonic bandgap fibers), the phase shift through the phase filters is not identical for all wavelengths in the selected wavelength range. This leads to a de-excitation point spread function (PSF) that is not ideal, and generally as a result to an undesirable reduction in fluorescence in the sharp maximum of the high-resolution PSF at the location of the geometric focus.
Phase filters are used in numerous procedures requiring increased resolution. This is particularly true for STED-, STED-4-pi-, and up-conversion-depletion microscopy. The phase filters change the phase of the wave front, penetrating them dependent on location. The standard method of manufacturing these phase filters is to apply structures to a glass substrate so that the wave front passing through experiences a location-dependent phase delay via the beam diameter. Because of this phase filter design, the phase shift that is generated is dependent on the wavelength of the light. However, this is generally not desirable because a different phase filter is needed for each wavelength used.
A scanning microscope that optically measures a sample point of a sample with high local resolution, with a light source to emit an excitation light beam adequate to excite an energy state in the sample, a detector to detect emission light, and a stimulation light beam emitted by a light source to generate stimulated emission in the sample point of the sample excited by the excitation light beam is known from DE 101 05 391 A1. The excitation light beam and the stimulation light beam are arranged such that their intensity distributions partially overlap each other in the focal region. The scanning microscope is characterized in that the optical elements that form the stimulation light beam are comprised into at least one module that can be positioned in the beam path of the scanning microscope. In one embodiment of the invention, means to influence the form of the focus of the stimulation light beam are envisaged.
A method similar to STED microscopy to increase resolution is known from T. Watanabe et. al., “Two-point-separation in super-resolution fluorescence microscope based on up-conversion fluorescence depletion technique,” 2003, Optics Express, Vol. 11, No. 24, 1 Dec. 2003. In so-called up-conversion-depletion microscopy, the excited state is not stimulated to de-excite as in the STED microscope; rather a further excitation into another state is brought about.